Sructural study of immuno regulatory proteins by NMR spectroscopy

作为逃避宿主免疫系统检测的策略的一部分，鼠巨细胞病毒（MCMV）编码三种调节I类主要组织相容性复合体（MHC-I）分子细胞表面表达的蛋白质：MHC-I同源物m152/gp 40，以及m02-m16家族成员m04/gp 34和m06/gp 48。先前对m04蛋白的研究揭示了一种趋异的免疫球蛋白（IG）样折叠，其是m02-m16家族的免疫evasins所特有的。在这里，我们工程和重组m06的特点，并研究其与全长和截短形式的MHC-I分子H2-Ld的几种技术的相互作用。此外，我们采用溶液NMR来映射MHC-I上的m06蛋白的相互作用足迹，利用截短的H2-Ld，mini-H2-Ld，仅由12个平台结构域组成。Mini-H2-Ld在体外与高亲和力肽重折叠，产生一个分子，显示出出色的NMR光谱特征，允许完整的骨架分配。这些基于NMR的研究揭示，m06与位于肽结合平台下的离散位点紧密结合，所述肽结合平台与MHC-I重链上的2-微球蛋白（2 m）界面部分重叠，这与体外结合实验一致，所述体外结合实验显示m06与2 m相关的MHC-I之间的复合物形成显著减少。此外，我们进行NMR弛豫实验来表征的ps-ns动力学的自由mini-H2-Ld MHC-I分子，揭示了相互作用的网站是高度有序的。这项研究提供了深入了解m06与MHC-I相互作用的机制，表明在肽加载途径的早期阶段对靶MHC-I分子进行了结构操纵。

As part of its strategy to evade detection by the host immune system, murine cytomegalovirus (MCMV) encodes three proteins that modulate cell surface expression of major histocompatibility complex class I (MHC-I) molecules: the MHC-I homolog m152/gp40, as well as the m02-m16 family members m04/gp34 and m06/gp48. Previous studies of the m04 protein revealed a divergent immunoglobulin (Ig)-like fold that is unique to immunoevasins of the m02-m16 family. Here, we engineer and characterize recombinant m06 and investigate its interactions with full length and truncated forms of the MHC-I molecule H2-Ld by several techniques. Furthermore, we employ solution NMR to map the interaction footprint of the m06 protein on MHC-I, taking advantage of a truncated H2-Ld, mini-H2-Ld, consisting of only the 12 platform domain. Mini-H2-Ld refolded in vitro with a high-affinity peptide to yields a molecule that shows outstanding NMR spectral features, permitting complete backbone assignments. These NMR-based studies reveal that m06 binds tightly to a discrete site located under the peptide-binding platform that partially overlaps with the 2-microglobulin (2m) interface on the MHC-I heavy chain, consistent with in vitro binding experiments showing significantly reduced complex formation between m06 and 2m-associated MHC-I. Moreover, we carry out NMR relaxation experiments to characterize the ps-ns dynamics of the free mini-H2-Ld MHC-I molecule, revealing that the site of interaction is highly ordered. This study provides insight into the mechanism of the interaction of m06 with MHC-I, suggesting a structural manipulation of the target MHC-I molecule at an early stage of the peptide-loading pathway.